BMP receptor 2 inhibition regulates mitochondrial bioenergetics to induce synergistic cell death with BCL-2 inhibitors in leukemia and NSLC cells

Background Bone morphogenetic protein (BMP) signaling cascade is a phylogenetically conserved stem cell regulator that is aberrantly expressed in non-small cell lung cancer (NSLC) and leukemias. BMP signaling negatively regulates mitochondrial bioenergetics in lung cancer cells. The impact of inhibiting BMP signaling on mitochondrial bioenergetics and the effect this has on the survival of NSLC and leukemia cells are not known. Methods Utilizing the BMP type 2 receptor (BMPR2) JL189, BMPR2 knockout (KO) in cancer cells, and BMP loss of function mutants in C elegans, we determined the effects of BMPR2 inhibition (BMPR2i) on TCA cycle metabolic intermediates, mitochondrial respiration, and the regulation of mitochondrial superoxide anion (SOA) and Ca++ levels. We also examined whether BMPR2i altered the threshold cancer therapeutics induce cell death in NSLC and leukemia cell lines. KO of the mitochondria uniporter (MCU) was used to determine the mechanism BMPR2i regulates the uptake of Ca++ into the mitochondria, mitochondrial bioenergetics, and cell death. Results BMPR2i increases mtCa++ levels and enhances mitochondrial bioenergetics in both NSLC and leukemia cell lines that is conserved in C elegans. BMPR2i induced increase in mtCa++ levels is regulated through the MCU, effecting mitochondria mass and cell survival. BMPR2i synergistically induced cell death when combined with BCL-2 inhibitors or microtubule targeting agents in both NSLC and leukemia cells. Cell death is caused by synergistic increase in mitochondrial ROS and Ca++ levels. BMPR2i enhances Ca++ uptake into the mitochondria induced by reactive oxygen species (ROS) produced by cancer therapeutics. Both acute myeloid leukemia (AML) and T-cell lymphoblastic leukemia cells lines were more responsive to the JL189 alone and when combined with venetoclax or navitoclax compared to NSLC.

Leukemias are more dependent on oxidative phosphorylation for ATP production compared to NSLC so could respond differently to BMPR2i.The effects of BMPR2i on the survival and mitochondrial bioenergetics in leukemia cells is not known.
We show here that BMPR2i increases mitochondrial bioenergetics, mtCa ++ and mtROS levels in both NSLC and leukemia cells.Our studies suggest that BMPR2i regulates mitochondrial redox sensing that promotes mtROS and mtCa ++ overload and cell death when combined with cancer therapeutics that increase ROS levels.Leukemia cells are more responsive to BMPR2 inhibition induced increase in mtCa ++ levels and cell death compared NSLC cells.Our studies suggest that a BMPR2 inhibitor may be effective in treating AML with p53 and MLL translocations, which are resistant to chemotherapeutics.

Cell viability analysis
Cells were seeded in 6-well plates and treated with selected agents.After the predetermined time point, live and dead cells were counted by a Vi-CELL BLU cell viability analyzer (Beckman Coulter Brea, CA, USA), which stains cells with trypan blue.The combination index (CI) was calculated using Compusyn software, and the IC 50 was calculated using GraphPad Prism.

Cell culture
The human cell lines H1299, Calu-1, and HEK-231 were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA).Jurkat and MDA-MB231 WT and MCU KO cells were generous gifts from Mohamed Trebak April of 2024 [19] (University of Pittsburgh) and Cristina Mammucari [20] (University of Padova, Italy), respectively.Jurkat and MDA-MB 231 cells were maintained in RPMI-1640 culture medium supplemented with 10% FBS.H1299, A549 WT, and A549 BMPR2 KO cells were maintained in low-glucose DMEM supplemented with 5% FBS, McCoy's medium supplemented with 10% FBS (Calu-1) and high-glucose DMEM supplemented with 10% FBS (HT-22) in a humidi ed incubator at 37°C with 5% CO 2 .All media were supplemented with 1× antibiotic and 1× L-glutamine.A549 BMPR2 KO and A549 WT cells with a 104 bp deletion of exon 11 in the kinase domain were purchased from FenicsBio (Helethorpe, MD) November 2023.HAP-1 WT and VDAC1 KO cells were purchased from Horizon Discovery on October 2023.HT-22 cells were purchased from Millipore October 2022 (October 2022).Cell lines were authenticated by suppliers and donors.

Metabolomics
LC − MS analysis of the cellular metabolites was performed on a Q Exactive PLUS hybrid quadrupoleorbitrap mass spectrometer (Thermo Scienti c) coupled to hydrophilic interaction chromatography (HILIC) as previously reported [21,22].

Immuno uorescence imaging
Immuno uorescence was performed as previously described [17].In brief, cells were plated onto sterile coverslips and treated for the designated time points.The cells were stained with MitoTracker Green for 30 minutes.The cells were washed and counterstained with DAPI.The cells were examined using a 60X oil lens and a uorescence microscope (Nikon Eclipse TE300).The studies were performed at least 4 times.

TMRM Staining
The cells were treated with DMSO (control), selected agents, and 50 µM CCCP (+ ve control) and subsequently stained with TMRM (Thermo-M20036) for 30 minutes at 37°C.After staining, the cells were washed with PBS, and after 30 minutes, the data were acquired on a Cytek Aurora ow cytometer.

Cytoplasmic and mitochondrial calcium analysis by ow cytometry
Mitochondrial and cytoplasmic calcium were analyzed using Rhod-2AM and Fluo-4AM staining, respectively.In brief, equal amounts of Flu-4AM or Rhod-2AM stock solution and the nonionic detergent Pluronic F-127 were diluted in media.The cells were stained for 45 minutes at 37°C.After staining, the cells were washed with PBS, and the data were acquired on a Cytek Aurora ow cytometer.

Apoptosis analysis by Annexin-FITC staining and ow cytometry
Apoptosis was analyzed by ow cytometry using a FITC Annexin V Apoptosis Detection Kit I (BD, Biosciences 559763) according to the manufacturer's protocol.In brief, cells were treated for the designated time points.After treatment, the cells were washed and stained with 100 µl of staining solution (5 µl of Annexin V-FITC and 5 µl of 7-AAD in 1X binding buffer).After staining, 400 µl of 1X binding buffer was added to each tube, and the cells were analyzed for apoptosis via ow cytometry (Cytek Aurora).

MitoTracker Green analysis by ow cytometry
The cells were treated with DMSO (control) and selected agents and subsequently stained with MitoTracker Green for 30 minutes at 37°C.After staining, the cells were washed with PBS, and the data were acquired on a Cytek Aurora ow cytometer.

Western blotting
Western blotting was performed as previously described [17].In brief, total protein was isolated by RIPA lysis buffer, subjected to SDS-PAGE and transferred to nitrocellulose membranes.The membrane was incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated secondary antibodies for 1 hour at room temperature.The band was visualized on X-ray lms.

Lung cancer xenograft
H1299 cells were mixed with 50% Matrigel in PBS, and 2x10 6 cells were injected into the anks of NCr nude mice (Taconic Biosciences).Tumors were isolated, and a single-cell suspension was prepared.A total of 1x10 6 cells were injected subcutaneously into the mice with 50% Matrigel.After visible tumor development, the mice were randomized into vehicle, JL189, ABT 263, and JL189 + ABT263 groups.
JL189 was dissolved in 5% NMP, 5% Solutol HS-15 and 90% citric acid, and ABT263 was dissolved in 30% PPG, 5% T80, 3.30% D5W, and 1% DMSO.JL189 (30 mg/kg twice a day) and ABT263 (12 mg/kg once a day) were injected by intraperitoneal (IP) injection 5 days a week for 3 weeks.Tumor size and volume were measured.Mice were euthanized for tumors exceeding 15 mm × 15 mm, pain, or loss of 20% body weight, as approved by Rutgers IACUC regulations, which were not met during the study.

Pharmacokinetics
The pharmacokinetic properties of JL189 were examined in BALB/c mice following intraperitoneal injection as previously described [23](Sai Life Science, Pune India).

Statistical analysis
A paired Student's t test, assuming unequal variances, was used to compare the means of the control with the mean of each treated group.Differences with p values < 0.05 were considered to indicate statistical signi cance.* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Results
BMPR2 inhibition increases TCA cycle intermediates.BMP2 ligand was previously shown to decrease TCA cycle intermediate in H1299 and A549 NSLC cell lines [17].We used JL189 a selective inhibitor of BMPR2 [17] to determine if BMP inhibition altered expression of TCA cycle intermediates in lung cancer cell lines.JL189 signi cantly increased the levels of the TCA cycle intermediates isocitrate, alphaketoglutarate, fumarate, malate, and ATP in H1299 cells (Fig. 1A).In A549 cells, JL189 signi cantly increased the expression of fumarate, malate and nicotinamide adenine dinucleotide (NADH) (Fig. 1A).
Western blot of A549 cells with kinase domain deletion of BMPR2 demonstrated signi cant decrease in ID1 compared to WT con rming downregulation of BMP signaling (Fig. 2E).A549 cells with BMPR2 KO had signi cantly greater levels of citrate, alpha-ketoglutarate, and ATP than A549 WT cells (Fig. 1B).To determine whether BMPR2i affects metabolism in noncancerous cells, metabolomic studies were performed on the HT-22 hippocampal neuronal mouse cell line.In HT-22 cells, JL189 decreased the levels of the glycolysis intermediates lactate and pyruvate while maintaining or increasing the levels of TCA cycle intermediates (Fig S1).The malate/pyruvate and α-ketoglutarate/pyruvate ratios increased, suggesting an increase in mitochondrial respiration (Fig S1).Both pharmacological and genetic inhibition of BMPR2 signaling showed similar effects on TCA cycle.BMPR2 inhibition increases oxidative phosphorylation.Inhibition of BMPR2 with JL189 in A549 cells signi cantly increased basal respiration, while spare respiratory capacity signi cantly increased in H1299 cells (Fig. 1C).A549 BMPR2-KO cells had signi cantly greater basal respiration, maximal respiration and spare respiratory capacity compared to A549 WT cells (Fig. 1D).Together, these data support that BMPR2i increases mitochondrial respiration in NSLC cell lines.BMPR2 inhibition increases mitochondrial mass.Dual immuno uorescence staining was performed for tubulin and the mitochondrial protein TFAM.Like prior studies using BMPR2 siRNA [24], inhibition of BMPR2 with JL189 destabilized the MT within 2 hr, and there was a clear change in the position of the mitochondria.In cells treated with JL189, the mitochondria moved from their typical perinuclear position to across the cytosol (Fig. 2A).By 24 hours, the intensity of TFAM uorescence was much greater in cells treated with JL189 (Fig. 2B), suggesting that there were more mitochondria.To quantify the mitochondrial mass, the cells were loaded with MitoTracker Green and examined by ow cytometry.

JL189 increased cytochrome b expression in H1299 and Jurkat cells (Fig G-H). The expression of cytochrome b was greater in A549
BMPR2-KO cells compared to A549 WT cells (Fig. 2I).These data suggest that BMPR2 inhibition increases mitochondrial bioenergetics in NSLC and leukemia cell lines.
BMPR2 inhibition increases mitochondrial calcium (mtCa ++ ) levels.The in ux of calcium into the mitochondria through the mitochondrial calcium uniporter (MCU) is a conserved mechanism that controls mitochondrial respiration, cell survival, and cell death when levels become too high [26,27].
Using the mitochondrial Ca ++ indicator Rhod2AM quanti ed by ow cytometry, we examined whether BMPR2i regulated mtCa ++ levels in NSLC and leukemia cells.JL189 signi cantly increased mtCa ++ levels in H1299, A549, and Calu-1 RAS mutated NSLC cell lines (Fig. 3A, C, G).There was also a signi cant increase in cytosolic Ca ++ levels (Fig B , D).The concentration of JL189 that increased mtCa ++ levels by 50% at 2 hr in H1299 cells was 0.84 µM (Fig. 3E).Immuno uorescence imaging con rmed that the increase in Rhod2AM uorescence occurred within the mitochondria and not within the cytosol (Fig. 3F).A549 BMPR2-KO cells had signi cantly greater mtCa ++ levels than did A549 WT cells, con rming that BMPR2 inhibition increases mtCa ++ levels (Fig. 3H).There was no difference in cytosolic Ca ++ levels between the A549 WT and A549 BMPR2 KO cells (Fig. 3H), suggesting that the increase in cytosolic Ca ++ levels induced by JL189 was a secondary event.JL189 also increased mtCa ++ levels in leukemia cell lines, Jurkat (T-ALL) and Kasumi1 (AML) and triple negative breast cancer (TNBC) cell line MDA 231 (Fig. 3I).
BMPR2 inhibition increases mtCa ++ levels in the absence of Ca ++ .Calu1 cells treated with JL189 in calcium-free media exhibited an increase in mtCa ++ levels within 20 seconds, which returned to baseline after 120 seconds (Fig. 3J).JL189 did not change cytosolic Ca ++ levels after 3 minutes (Fig. 3K,M).
Thapsigargin, which blocks endoplasmic Ca ++ -ATPase (SERCA) and leads to an increase in cytosolic Ca ++ , was used as a positive control (Fig. 3L-M).These studies indicate that the increase in mtCa ++ following BMPR2 inhibition is not caused by an increase in the uptake of extracellular calcium.The studies also suggest that the increase in mtCa ++ levels is not from an increase in cytosolic Ca ++ levels released from calcium storage organelles.
BMP inhibition in C. elegans increases mtCa ++ levels and mitochondrial mass.To determine whether BMP regulation of mitochondrial calcium and mitochondrial mass is conserved, we utilized C. elegans harboring BMP ligand (dbl-1) loss-of-function (lof) mutants and the red uorescent mitochondrial calcium sensor LAR-GECO [28] under the control of the myo-3 promoter [29].Worms were also crossed to generate animals that expressed the green uorescent protein (GFP) transgene under the control of the myo-3 promoter, which localizes to the mitochondria (MitoGFP) and nucleus.MitoGFP was used to determine mitochondrial mass, and the LAR-GECO uorescence intensity was normalized to MitoGFP to determine basal mtCa ++ levels.Compared with those of the WT, the animals harboring the dbl-1 lof transgene had signi cantly greater mtCa ++ levels (Fig. 4A-B) and greater mitochondrial mass (Fig. 4A, C).
BMPR2i synergistically enhances cell death when combined with BCL-2 inhibitors.We hypothesized that increasing mtCa ++ levels would increase oxidative stress enhancing mitochondrial-induced cell death by cancer therapeutics.BCL-2 inhibitors were studied rst since they mediate mitochondrial-induced cell death.ABT-263 (Navitoclax) inhibits BCL-2 and BCL-xL and ABT-199 (Venetoclax) is speci c for BCL-2.Cell counts were performed using a ViBlue cell counter, which stains cells with trypan blue to determine cell death.Synergy was determined by calculating the combination index [30].JL189 combined with ABT-263 synergistically induced cell death in both NSLC and leukemia cell lines (Fig. 5A-B).In total, we found that JL189 combined with ABT-263 synergistically induced cell death in 4 NSCLCs, 2 acute lymphoblastic leukemia (T-ALL) cell lines (Jurkat and DNT-41), 2 acute myelogenous leukemia (AML) cell lines (Kasumi1, THP-1) (Table S1).BMPR2i with JL189 alone and when combined with ABT-263 induced more cell death in leukemia cell lines (Fig. 5B) compared to NSLC cell lines (Fig. 5A), despite using a lower dose of ABT-263.In leukemia cells, JL189 in combination with ABT-199 (Venetoclax) induced synergistic cell death with little response in NSLC cells (Fig S2).The immortalized human embryonic kidney cells (HEK292) were not responsive to the combination of JL189 and ABT-263 (Fig. 5C), suggesting that cancer cells are more susceptible to cell death induced by this combination.
BMPR2i combined with ABT-263 induces mitochondria-induced cell death.In leukemia and NSCLC cell lines, the combination of JL189 with ABT-263 after 5 hr induced a much greater increase in the expression of activated caspase-3 fragment and cleavage of PARP-1 compared to each compound alone (Fig. 5D).Jurkat cells exhibited a signi cant decrease in the mitochondrial membrane potential (MMP) after 2 hr when JL189 was combined with ABT-263 but not with either compound alone (Fig. 5E).The MMP of a mouse hippocampal neuronal cell line (HT-22 cells) did not decrease after treatment with JL189 combined with ABT-263 (Fig. 5F).In H1299 cells, the combination of JL189 and ABT-263 signi cantly increased MMP compared to each compound alone (Fig. 5G).Hyperpolarization of the mitochondrial can occur when electron transport is dysfunctional.Mitochondrial respiration in H1299 cells was signi cantly decreased after treatment with the combination of JL189 and ABT-263 compared to that after each treatment alone (Fig. 5H), demonstrating mitochondrial dysfunction.These studies suggest that the combination of JL189 and ABT-263 promotes mitochondrial dysfunction to induce cell death.
BMPR2i synergistically enhances cell death when combined with microtubule-targeting drugs.We examined whether BMPR2i synergizes with commonly used chemotherapeutics that target microtubules.When used in combination with JL189, vincristine synergistically induced cell death in the T-ALL, AML, and CML HAP-1 cell lines (Table S1).Synergistic cell death also occurred in K-Ras-mutated NSCLC cell lines treated with JL189 and Taxol (Table S1).To con rm that BMPR2i is required for the observed synergistic cell death, A549 WT and BMPR2 KO cells were examined.Both Taxol and vinblastine induced signi cantly more cell death in the A549 BMPR2-KO cells than in the A549 WT cells (Fig. 5I).
BMPR2 inhibition combined with ABT-263 synergistically increases mtCa ++ levels.Ca ++ enhances mitochondrial respiration by regulating three TCA cycle dehydrogenases, thereby directly controlling ATP synthesis [31].However, when mtCa ++ levels become too high, mitochondria-induced cell death is triggered [32,33].Since BCL-2/BCL-xL inhibit the in ux of Ca ++ into mitochondria [34], we examined whether the combination of JL189 and ABT-263 synergistically increased mtCa ++ levels.The cells were loaded with Rhod2AM and DAPI to exclude dead cells from the analysis.Signi cantly greater mtCa ++ levels were detected in H1299, A549, Jurkat, and MDA-231 cells treated with the combination of JL189 and ABT-263 than in those treated with each compound alone (Fig. 6A, C, D, E).When calcium was removed from the cell culture medium, the combination of JL189 with ABT-263 still induced the highest mtCa ++ levels (Fig. 6B, E).A549 BMPR2 KO cells did not show increased mtCa ++ levels in response to JL189 alone or in combination with ABT-263 (Fig. 6F).Synergistic cell death induced by JL189 combined with ABT-263 also occurred in the presence or absence of Ca ++ (Fig. 6G).These studies demonstrate signi cantly increased mtCa ++ levels induced by BMPR2 inhibition combined with ABT-263, which is associated with cancer cell death.These studies also show that cell death and elevated mtCa ++ levels are not dependent on the in ux of extracellular Ca ++ .BMPR2 inhibition combined with Taxol synergistically increases mtCa ++ levels.A synergistic increase in mtCa ++ also occurred in Calu1 and H1299 cells treated with JL189 combined with Taxol (Fig. 6H, I).MTtargeting agents are not known to increase mtCa ++ levels.However, MT-targeting agents increase reactive oxygen species (ROS), which can increase mtCa ++ uptake through the mitochondrial uniporter (MCU) [35].This raised the question of whether MT-targeting agents and BCL-2 inhibitors increase mtCa ++ levels by increasing ROS levels.
Increasing free radicals increase mtCa ++ levels [36], which can amplify the increase in mtCa ++ and ROS levels [36].The free radical scavenger vitamin E was used to determine the role of ROS in regulating mtCa ++ levels and cell death.Vitamin E effectively decreased the increase in total O2•-(Fig.7D) and mtO2•-(Fig.7E) levels in cells treated with JL189 combined with ABT-263.Vitamin E also signi cantly decreased the increase in mtCa ++ levels in Calu-1 cells (Fig. 7F) and decreased cell death in Calu1-1, A549, H1299, and Jurkat cells induced by JL189 combined with ABT-263 (Fig. 7G-J).These studies show that the combination of JL189 with ABT-263 induces high ROS levels, which further increases mtCa ++ levels leading to cell death.BMPR2 KO increases O2•-levels.Compared with WT cells, A549 BMPR2-KO cells expressed more total O2•-(Fig.7K).Compared with WT cells, BMPR2 KO cells exhibited greater increases in O2•-levels when treated with cisplatin (Fig. 7L).Treatment with vitamin E signi cantly decreased the death of BMPR2-KO cells treated with cisplatin (Fig. 7M).BMPR2 KO studies con rmed that BMPR2i increases O2•-levels.These studies also validate that BMPR2i primes cancer cells for further increases in ROS production when they are challenged with a cancer therapeutic, which induces cell death.
Taxol increases O2• -levels, leading to increased mtCa ++ levels.We next asked whether the chemotherapeutic Taxol, that used to treat NSLC, also induces an increase in mtROS levels, which regulates an increase in mtCa ++ levels.Taxol increased mtO2•-levels in Calu1 cells (Fig. 7N).The combination of JL189 and Taxol increased mtO2•-to levels greater than those induced by either compound alone (Fig. 7N).Vitamin E effectively attenuated the increase in mtO2•-induced by JL189 combined with Taxol (Fig. 7O).Vitamin E decreased the increase in mtCa ++ levels induced by Taxol (Fig. 7P).These studies demonstrate that Taxol and potentially other chemotherapeutic agents increase ROS levels that promote Ca ++ uptake into the mitochondria.These studies suggest that BMPR2 inhibition synergizes with cancer therapeutics by inducing high ROS levels, which ampli es the in ux of calcium into the mitochondria, leading to mtROS and mtCa ++ overload.
The increase in mtCa ++ and mtROS induced by JL189 and ABT-263 is greater in Jurkat cells compared to H1299 cells.To better understand why leukemia cells are more sensitive to cell death, we compared Jurkat and H1299 cells using the same dose of JL189 and ABT-263.Because cell death was so high, we used a lower dose of JL189 and ABT-263 leukemia cells compared to the NSLC cell lines.At the same dose, JL189 caused a greater increase in mtCa ++ levels after 2 hr in Jurkat cells compared to H1299 cells (Fig. 6Q).ABT-263 caused a signi cantly greater increase in mtO2•levels in Jurkat compared to H1299 cells (Fig. 6R).These data suggest that leukemia cells are more sensitive to BMPR2i induced increase in mtCa ++ levels and BCL-2 inhibition increase in mtROS levels compared to NSLC cells.
BMPR2i does not regulate NADPH oxidase to induce cell death.O2•-can also be produced by NADPH oxidase in the cytosol [36].NADPH oxidase activity is inhibited by apocynin.Apocynin did not decrease the cell death induced by JL189 combined with ABT-263 in either A549 or Calu1 cells (Fig S3A -B).Glutathione is the major free radical scavenger in a cell.If BMPR2i induces oxidative stress in the cytosol, then depleting glutathione should synergize with JL189.L-Buthionine sulfoximine (BSO) is an inhibitor of g-glutamylcysteine synthetase and depletes glutathione levels.BSO did not increase the degree of cell death induced by JL189 in A549 or Calu1 cells (Fig S3C -D).These ndings support our other studies demonstrating that mitochondria are the source of ROS that induces cell death in cells treated with JL189 and ABT-263.
BMP signaling regulates the in ux of Ca ++ into the mitochondria induced by ROS.After 2 hr of treatment with JL189, the baseline mtCa ++ levels remained elevated (Fig. 8A-B).When cells were pretreated with vitamin E, JL189 did not increase basal mtCa ++ levels (Fig. 8B).These data suggest that the sustained increase in mtCa ++ levels after treatment with JL189 is mediated by an increase in ROS levels.JL189 did not increase mtO2• levels during the rst 3 minutes after treatment in Calu1 cells (Fig. 8C), suggesting that the initial increase in mtCa ++ levels is not dependent on an increase in ROS.Hydrogen peroxide (H2O2) rapidly increased mtCa ++ levels, which were enhanced by JL189 in Calu1 cells (Fig. 8D).
Conversely, the addition of BMP2 ligand decreased the increase in mtCa ++ levels induced by H2O2 (Fig. 8E).These studies suggest that BMP signaling regulates the in ux of calcium into the mitochondria induced by ROS.BMPR2 inhibition does not regulate lysosomal or endoplasmic reticulum (ER) Ca ++ stores.Lysosomes and the ER directly transfer Ca ++ into mitochondria through voltage-dependent anion channels (VDAC) [37].Under Ca ++ -free conditions, if JL189 induced the transfer of Ca ++ from either the ER or lysosomes, the remaining stored Ca ++ would be decreased.Thapsigargin was used to deplete the remaining ER Ca ++ stores.ML-SA1, which activates the lysosome e ux receptor TRPML-1 [38], was used to deplete lysosomal Ca ++ stores.H1299 cells treated with JL189 for 16 hr in media without Ca ++ did not signi cantly deplete ER or lysosomal Ca ++ stores (Fig S4A -B).Chronic myelogenous leukemia HAP-1 VDAC1 KO cells have a 14 bp deletion in exon 6. VDAC1 knockout did not signi cantly attenuate the increase in mtCa ++ levels after treatment with JL189 for 2 hr or 4 hr (Fig S5A -B).VDAC1 KO did not affect the mitochondrial mass or cell growth of cells treated with JL189 (Fig S5C -D).These studies suggest that BMPR2i does not regulate the ER or lysosomes to increase mtCa ++ levels.
BMPR2 inhibition-induced increases in mtCa ++ levels, mitochondrial mass, and cell death are dependent on the mitochondrial uniporter (MCU).MCU regulates the rapid entry of cytosolic Ca ++ into the mitochondrial matrix [39].To test whether BMP signaling mediates Ca ++ uptake through MCU, we utilized Jurkat and MDA 231 cells with MCU KO via CRISPR-Cas9 [19,20].Western blot analysis con rmed the KO of MCU (Fig. 8F).Compared with control cells, Jurkat WT cells treated with JL189 for 16 hr had a 37% greater increase in mtCa ++ levels (Fig. 8G).Compared with control cells, Jurkat MCU KO cells exhibited only a 10% greater increase in mtCa ++ (Fig. 8G).JL189 caused a 49% increase in mtCa ++ levels in MDA 231 WT cells compared with a 16% increase in mtCa ++ levels in MDA 231 MCU KO clone 1 cells and a 28% increase in mtCa ++ levels in clone 2 cells after 5 hr (Fig. 8H).These studies suggest that the in ux of Ca ++ into the mitochondria induced by BMPR2i is regulated through the MCU.
JL189 increased the uorescence intensity of MitoTracker Green by 18% in the MDA-231 WT cells and increased it by only 4% in the MDA-231 MCU KO cells (Fig. 8I).JL189-induced cell death was signi cantly greater in Jurkat WT cells than in MCU KO cells after 24 and 48 hr (Fig. 8J).JL189 induced more cell death in the MDA 231 WT cells than in the MCU KO cells (Fig. 8K).Compared with that in WT cells, growth suppression induced by the combination of JL189 and ABT-263 was partially suppressed in MDA-231 MCU KO cells (Fig. 8L).These data suggest that the increase in mitochondrial bioenergetics and cell death induced by BMPR2i are mediated by an increase in the in ux of Ca ++ through the MCU.
Treatment with JL189 combined with ABT-263 synergistically decreased the growth of lung tumor xenografts in mice.Since JL189 and ABT-263 when used alone induce minimal cell death in H1299 cells in vitro, we examined synergy in tumor xenografts in mice using H1299 cells.JL189 or ABT-263 alone had no effect on tumor growth of H1299 tumor xenografts.There was an approximately a 50% greater reduction in the tumor weight in mice treated with the combination of JL189 and ABT-263 compared to mice treated with vehicle or each compound alone (Fig. 9A-C).Pharmacokinetic studies demonstrated that JL189 has a serum half-life of only 60 minutes in mice (Table S2).Even with twice daily dosing, the therapeutic window is estimated to be only 4 hr.This study supports that the combination of JL189 combined ABT-263 induced synergy in lung tumor xenografts despite a short therapeutic window in mice.
BMPR2 inhibition enhances mitochondrial bioenergetics in lung tumor xenografts in mice.Compared with those treated with vehicle, tumors treated with JL189 alone or in combination with ABT-263 had signi cantly greater expression of mtATP and cytochrome c (Fig. 9D-E).The combination of JL189 and ABT-263 had signi cantly higher expression cytochrome b that trended toward signi cance with JL189 alone (Fig. 9D-E).mtATP and cytochrome b are transcribed from mitochondrial DNA [25,40].These data suggest that BMPR2 inhibition increases mitochondrial bioenergetics in lung tumor xenografts.

Discussion
Although BMP signaling is the oldest conserved signaling pathways in metazoans, its role in regulating energy homeostasis has only recently been realized [10].Our prior reports suggested that BMP signaling negatively affects energy homeostasis, which may have implications in cancer and other age-related diseases [10].In this study, we show that BMPR2i improves mitochondrial bioenergetics, as demonstrated by an increase in TCA cycle intermediates, oxidative phosphorylation, and mitochondrial mass.An improvement in mitochondrial bioenergetics was shown not only in cancer cells but also in normal mouse hippocampal neurons and in C. elegans, suggesting that the regulation of energy homeostasis by BMPR2 is conserved.
The regulation of ROS levels is a conserved mechanism determining stem cell self-renewal and the initiation of differentiation [41].During self-renewal, stem cells have low respiratory capacity and are highly dependent on glycolysis, similar to the Warburg effect in cancer cells [41,42].Although BMP signaling is a critical regulator of self-renewal and cell fate decisions, it has not been previously shown to regulate ROS levels.Interestingly, in cancer cells, BMP signaling regulates mitochondrial ROS and Ca ++ levels, and the regulation of mtCa ++ levels is conserved in C. elegans.Our studies suggest that BMPR2i regulates MCU to increase mtCa ++ levels.The increase in mtCa ++ levels regulated mitochondrial mass, and some of the cell death induced by BMPR2i alone.
Since cancer cells are already under oxidative stress, they are more susceptible to additional increases in ROS levels [43][44][45].BMPR2i alone may have bene cial effects on mitochondrial bioenergetics in normal cells as suggested in HT-22 cells.However, in cancer cells, the increased oxidative stress induced by BMPR2i appears to prime cells for cell death.Our studies suggest that BMPR2i enhances then in ux of calcium into the mitochondria induced by mtROS produced by cancer therapeutics leading the mtROS and mtCa ++ overload and cell death.ROS and mtCa ++ are essential for mitochondrial function, but when they are too high, as in our studies, cell death is induced through conserved apoptotic and nonapoptotic mitochondria-induced cell death pathways [36,46].Interestingly, synergistically cell death occurred in cell lines with different genetic mutations.These studies suggest that BMPR2i mediated cell death pathways are not dependent on a speci c genetic mutation.
Although the comparison between leukemia and NSLC was not complete, we provide evidence that BMPR2i improves some bioenergetic properties in both leukemia and NSLC cells, which involves the in ux of Ca ++ into the mitochondria.The mitochondria in cancer cells often reprogram metabolic pathways to promote survival and chemoresistance.Adaptive mechanisms include mitochondrial tra cking, Ca ++ transfer, ROS signaling, mtDNA synthesis, and mitochondrial ssion [47].This has led to several approaches to limit adaptive changes that include targeting mitochondrial complexes (I-V), TCA cycle, redox balance, and metabolic pathways [47].Our studies suggest the BMPR2i regulates redox sensing of the MCU to alter the balance of ROS and mtCa ++ levels when combined with certain cancer therapeutics.The mechanism by which BMPR2i regulates the MCU needs to be elucidated to better understand how to implement this strategy to treat patient.
Our studies show that BMPR2i with J189 induced higher levels of mtCa ++ levels and cell death in leukemia cell lines compared to NSLC cell lines.JL189 combined with either ABT-263 (navitoclax) or ABT-199 (venetoclax) induced synergistic cell death in AML and T-ALL leukemia cell lines that was much greater compared to NSLC cell lines.Venetoclax combined with a hypomethylating agent is the rst line treatment for AML patients over 75 and for patients who cannot tolerate intensive chemotherapy.The prognosis with the combination of venetoclax and hypomethylating agent is poor with a median survival of only 14.7 months.Importantly, there is no effective treatment of AML with p53 mutation or mixed lineage leukemia (MLL) translocations [48,49].Kasumi-1 cells have a p53 mutation and THP-1 cells have a p53 and MLL-AF9 translocation.We show that Kasumi-1 and THP-1 are very responsive to JL189 alone and in combination with venetoclax, suggesting that a BMPR2 inhibitor could be effective in treating AML.

Conclusions
Our studies suggest that BMPR2i regulates the MCU to increase mitochondrial Ca ++ levels, which mediate mitochondrial mass and cell death.BMPR2i enhances the in ux of Ca ++ into the mitochondria induced by ROS.When BMPR2i is combined with chemotherapeutics it increases mtCa ++ and mtROS levels inducing a metabolic switch that initiates conserved mitochondrial cell death pathways.BMPR2i represents a novel approach to induce synergistic cell death by regulating mitochondrial Ca ++ and ROS levels.Our studies support the continued development of BMPR2 inhibitors into a drug and their evaluation in patients with AML.

Supplementary Files
This is a list of supplementary les associated with this preprint.Click to download. FigureS1.tif